Bottom Line:
In addition, a fourfold decrease in the number of HSCs could be demonstrated in a competitive repopulating assay.Secondary transplantations of marrow cells from primary recipients of p55(-/-) marrow revealed impaired self-renewal ability of p55-deficient HSCs.These data show that, in vivo, signaling through the p55 subunit of the TNF receptor is essential for regulating hematopoiesis at the stem cell level.

ABSTRACTHematopoietic stem cell (HSC) self-renewal is a complicated process, and its regulatory mechanisms are poorly understood. Previous studies have identified tumor necrosis factor (TNF)-alpha as a pleiotropic cytokine, which, among other actions, prevents various hematopoietic progenitor cells from proliferating and differentiating in vitro. However, its role in regulating long-term repopulating HSCs in vivo has not been investigated. In this study, mice deficient for the p55 or the p75 subunit of the TNF receptor were analyzed in a variety of hematopoietic progenitor and stem cell assays. In older p55(-/-) mice (>6 mo), we identified significant differences in their hematopoietic system compared with age-matched p75(-/-) or wild-type counterparts. Increased marrow cellularity and increased numbers of myeloid and erythroid colony-forming progenitor cells (CFCs), paralleled by elevated peripheral blood cell counts, were found in p55-deficient mice. In contrast to the increased myeloid compartment, pre-B CFCs were deficient in older p55(-/-) mice. In addition, a fourfold decrease in the number of HSCs could be demonstrated in a competitive repopulating assay. Secondary transplantations of marrow cells from primary recipients of p55(-/-) marrow revealed impaired self-renewal ability of p55-deficient HSCs. These data show that, in vivo, signaling through the p55 subunit of the TNF receptor is essential for regulating hematopoiesis at the stem cell level.

Figure 4: Purification strategy for isolating Sca-1+Lin−/loc-Kit++ CD34− cells. Cells that express Sca-1 were identified as in the legend to Fig. 2. Depicted are representative profiles of viable Sca-1+ WT marrow cells after lineage depletion. The Sca-1+c-Kit++ cells, indicated by quadrants 1 and 2, showed no or low levels of mature Lin expression (A). Thus, identifying Lin markers as a means to further purify “Lin-depleted” Sca-1+c-Kit++ cells is not very helpful. In contrast, replacing the mAb cocktail directed against the Lin markers with an anti-CD34 mAb allows the identification of a small subpopulation of the Sca-1+Lin−/loc-Kit++ cells (∼10%), the CD34− cells, that has been shown to include the long-term repopulating HSCs (B, quadrant 3) (reference 43).

Mentions:
One possible explanation for the observed differences in HSC number and proliferative potential between WT and p55−/− mice is a higher cycling rate of p55−/− HSCs. Purified Sca-1+Lin−/loc-Kit++ cells still include large numbers of committed progenitor cells, as confirmed by our finding that between 20 and 30% of these cells (WT and knockout cells) were in the S/G2/M phase of the cell cycle (data not shown). Therefore, we further subdivided this population using an mAb against the CD34 antigen. The Sca-1+Lin−/loc-Kit++CD34− cells, which include the long-term reconstituting HSCs 43, comprise ∼10% of all Sca-1+Lin−/loc-Kit++ cells. The sorting strategy for obtaining these cells is depicted in Fig. 4. The results of cell cycle analyses of unseparated BM cells and highly purified cells (box 3) are shown in Table . As expected, DNA staining of unseparated BM cells did not demonstrate a difference in cycling activity between the WT and either knockout mouse. Moreover, the result obtained with the highly purified CD34− subpopulation of Sca-1+Lin−/loc-Kit++ cells isolated from p55−/− marrow cells did not strongly suggest that cycling activity of p55−/− HSCs was altered. Therefore, a difference in percentage of cycling HSCs does not seem to explain the observed qualitative and quantitative differences between HSCs from WT and p55-deficient mice.

Figure 4: Purification strategy for isolating Sca-1+Lin−/loc-Kit++ CD34− cells. Cells that express Sca-1 were identified as in the legend to Fig. 2. Depicted are representative profiles of viable Sca-1+ WT marrow cells after lineage depletion. The Sca-1+c-Kit++ cells, indicated by quadrants 1 and 2, showed no or low levels of mature Lin expression (A). Thus, identifying Lin markers as a means to further purify “Lin-depleted” Sca-1+c-Kit++ cells is not very helpful. In contrast, replacing the mAb cocktail directed against the Lin markers with an anti-CD34 mAb allows the identification of a small subpopulation of the Sca-1+Lin−/loc-Kit++ cells (∼10%), the CD34− cells, that has been shown to include the long-term repopulating HSCs (B, quadrant 3) (reference 43).

Mentions:
One possible explanation for the observed differences in HSC number and proliferative potential between WT and p55−/− mice is a higher cycling rate of p55−/− HSCs. Purified Sca-1+Lin−/loc-Kit++ cells still include large numbers of committed progenitor cells, as confirmed by our finding that between 20 and 30% of these cells (WT and knockout cells) were in the S/G2/M phase of the cell cycle (data not shown). Therefore, we further subdivided this population using an mAb against the CD34 antigen. The Sca-1+Lin−/loc-Kit++CD34− cells, which include the long-term reconstituting HSCs 43, comprise ∼10% of all Sca-1+Lin−/loc-Kit++ cells. The sorting strategy for obtaining these cells is depicted in Fig. 4. The results of cell cycle analyses of unseparated BM cells and highly purified cells (box 3) are shown in Table . As expected, DNA staining of unseparated BM cells did not demonstrate a difference in cycling activity between the WT and either knockout mouse. Moreover, the result obtained with the highly purified CD34− subpopulation of Sca-1+Lin−/loc-Kit++ cells isolated from p55−/− marrow cells did not strongly suggest that cycling activity of p55−/− HSCs was altered. Therefore, a difference in percentage of cycling HSCs does not seem to explain the observed qualitative and quantitative differences between HSCs from WT and p55-deficient mice.

Bottom Line:
In addition, a fourfold decrease in the number of HSCs could be demonstrated in a competitive repopulating assay.Secondary transplantations of marrow cells from primary recipients of p55(-/-) marrow revealed impaired self-renewal ability of p55-deficient HSCs.These data show that, in vivo, signaling through the p55 subunit of the TNF receptor is essential for regulating hematopoiesis at the stem cell level.

ABSTRACTHematopoietic stem cell (HSC) self-renewal is a complicated process, and its regulatory mechanisms are poorly understood. Previous studies have identified tumor necrosis factor (TNF)-alpha as a pleiotropic cytokine, which, among other actions, prevents various hematopoietic progenitor cells from proliferating and differentiating in vitro. However, its role in regulating long-term repopulating HSCs in vivo has not been investigated. In this study, mice deficient for the p55 or the p75 subunit of the TNF receptor were analyzed in a variety of hematopoietic progenitor and stem cell assays. In older p55(-/-) mice (>6 mo), we identified significant differences in their hematopoietic system compared with age-matched p75(-/-) or wild-type counterparts. Increased marrow cellularity and increased numbers of myeloid and erythroid colony-forming progenitor cells (CFCs), paralleled by elevated peripheral blood cell counts, were found in p55-deficient mice. In contrast to the increased myeloid compartment, pre-B CFCs were deficient in older p55(-/-) mice. In addition, a fourfold decrease in the number of HSCs could be demonstrated in a competitive repopulating assay. Secondary transplantations of marrow cells from primary recipients of p55(-/-) marrow revealed impaired self-renewal ability of p55-deficient HSCs. These data show that, in vivo, signaling through the p55 subunit of the TNF receptor is essential for regulating hematopoiesis at the stem cell level.